Collagenolytic activity from venom of the rattlesnake Crotalus atrox.

Abstract

A specific collagenase from a vertebrate source was first demonstrated by Gross and Lapiere (1). Collagenase was subsequently found in the media from tissue cultures of a variety of normal animal tissues (2-7) as well as several human and animal tumors (8). Delaunay and co-workers (9) reported that solutions of Vipera aspis venom contained weak collagenolytic activity and Hadidian reported that venom from Agkistrodon piscivorus is known to liquefy gelatin (10). We have demonstrated that venom from four species of snakes contains collagenolytic activity. Also, venom from Crotalus atrox degrades mesenteric native collagen fibers during tissue cultures (11). This report describes further evidence that the collagenolytic activity from Crotalus atrox venom is due to a specific collagenase. Materials and Methods. Lyophilized venom from Crotalus atrox was purchased from Sigma Chemical Company, St. Louis, Missouri. Rat-tail collagen solutions were prepared by extraction with acetic acid according to the procedure of Bornstein (12). Three times reconstituted rat-skin collagens were prepared by the method of Piez, et al. (13). The purity and native state of collagen was established by resistance to trypsin digestion. Collagenase activity was determined by viscometry. Viscosity changes were measured in Ostwald viscosimeters with flow times for water ranging between 77 and 90 sec at 27°. The reaction mixture had a final volume of 7.0 ml and contained 3 mM Tris-HCl, pH 7.0, 3 mM CaCl2, rat-tail collagen adjusted to a final specific viscosity of approximately 8.0 or rat-skin collagen with a final collagen concentration of 0.2%, and venom at various concentrations. Samples to be electrophoresed were subjected to thermal denaturation at 45° for 10 min (15). Disc electrophoresis as modified by Reisfeld, et al. (16) for basic proteins and revised by Nagai (15) was used.